Production of weakly active or inactive mutants of alkaline phosphatase and their expression in yeast

ABSTRACT

The invention concerns a method for the recombinant production or expression of eukaryotic alkaline phosphatase mutants in yeast cells wherein the specifically introduced mutations result in a reduction of the specific AP activity by at least a factor 1:100. The invention also concerns a method for inserting corresponding nucleic acid sequences into a vector for expression in methylotrophic yeast strains and it concerns corresponding vectors and host strains.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/395,790filed Mar. 24, 2003 and claims priority to German application DE10213201.1 filed Mar. 25, 2002.

FIELD OF THE INVENTION

The invention concerns a method for the recombinant production andexpression of mutants of a eukaryotic alkaline phosphatase which isweakly active or inactive. In addition the invention concernscodon-optimized DNAs based on the nucleic acid sequence which codes fora highly-active alkaline phosphatase and which has been modified bydirected mutagenesis in such a manner that it codes for an alkalinephosphatase which has only a weak activity or is inactive. The inventionalso concerns a method for inserting the mutated DNA into a vector forexpression in yeast cells and a method for expressing the alkalinephosphatase mutants in yeast.

BACKGROUND

Alkaline phosphatases (AP) are dimeric, zinc-containing, non-specificphosphomono-esterases which occur in prokaryotic and eukaryoticorganisms e.g. in E. coli and mammals (McComb et al., 1979 AlkalinePhosphatases Plenum Press, New York). A comparison of the primarystructure of various alkaline phosphatases showed that there is a highdegree of homology (25-30% homology between E. coli and mammalian AP;Millàn, 1988 Anticancer Res. 8, 995-1004; Harris, 1989 Clin. Chim. Acta186, 133-150).

In humans and higher animals the AP family consists of four memberswhich are coded in different gene loci (Millàn, 1988 Anticancer Res. 8,995-1004; Harris 1989 Clin. Chim. Acta 186, 133-150). The family ofalkaline phosphatases includes the tissue-specific APs (placental AP(PLAP), germ cell AP (GCAP) and intestinal AP (IAP)) and thenon-tissue-specific APs (TnAP) which are mainly located in the liver,kidney and bones.

An important property of the previously known APs is the largevariability in the catalytic activity of mammalian APs which have a10-100-fold higher k_(cat)s value than E. coli AP. Among the mammalianAPs the APs from the bovine intestine (bIAP) exhibit the highestspecific activities. This property makes the bIAPs attractive forbiochemical applications such as e.g. the use of corresponding enzymeconjugates as a diagnostic reagent or for dephosphorylating DNA. Theexistence of various alkaline phosphatases from the bovine intestinewhich have different levels of specific activity is described in EP 0955369 and Manes et al. (1998), J. Biol. Chem. 273 No. 36, 23353-23360. Upto now recombinant expression of eukaryotic alkaline phosphatases of lowactivity (up to 3000 U/mg) has been described in various eukaryotic celllines such as CHO cells (bIAP I/WO 93/18139; Weissig et al. 1993,Biochem. J. 260, 503-508), COS cells (human placental AP/Berger et al.1987 Biochemistry 84, 4885-4889) or baculovirus expression system (humanplacental AP/Davis et al. 1992, Biotechnology 10, 1148-1150). Theexpression of APs having a higher activity (spec. activity>3000 U/mg)from the bovine intestine in CHO cells has also been described (bIAP II,III and IV/Manes et al. 1998, J. Biol. Chem. 273 No. 36, 23353-23360).However, a disadvantage of expressing alkaline phosphatases in theseexpression systems is the low expression rate which makes ituneconomical to produce eukaryotic alkaline phosphatase recombinantly.

Although in principle it is possible to express eukaryotic alkalinephosphatases in prokaryotic expression hosts such as E. coli (humanplacental AP/Beck and Burtscher, 1994 Protein Expression andPurification 5, 192-197), the alkaline phosphatases expressed inprokaryotes have no glycosylation which is essential especially for thepreparation of enzyme conjugates depending on the conjugate method.

Alkaline phosphatase is often used as an enzyme conjugate in the form ofa complex with an antibody. In this case the alkaline phosphatase isconjugated with an antibody which is directed against a particularantigen. This antigen is firstly bound in a first reaction by anantibody immobilized on a vessel wall which recognizes a differentepitope on the target antigen than does the antibody-AP conjugate. Thisantibody-antigen complex is then detected in a second reaction by thebinding of the antibody-AP conjugate. False-positive results occurrepeatedly in such tests and are caused by an unspecific binding of theantibody-AP conjugate to the vessel wall or to the first antibody. Theseinterferences can be prevented by adding an excess of a conjugatecontaining an inactive or weakly active AP mutant as aninterference-eliminating protein. However, in order to act veryspecifically as an interference-eliminating protein, the AP mutant must,in addition to having a low activity or no activity, also haveessentially the same tertiary and quaternary structure.

Hence the object of the present invention is to use directed mutagenesisto produce mutants of alkaline phosphatase as aninterference-eliminating protein which are only very weakly-active orcompletely inactive but whose amino acid sequence is only slightlymodified and have a tertiary and quaternary structure that is changed aslittle as possible. Another object of the invention is to develop arobust and stable expression method for producing glycosylatedeukaryotic alkaline phosphatase mutants which also enables an economicalproduction of a corresponding alkaline phosphatase mutant due to thehigh expression rate.

SUMMARY OF THE INVENTION

The present invention concerns mutants of eukaryotic alkalinephosphatase wherein the sequence to be mutated is at least 77%homologous to SEQ ID NO: 2, the mutant has an activity which is reducedby at least 100-fold compared to the wild-type, and the mutant has oneor more of the following mutations, with the position of the mutationdefined relative to the position in SEQ ID NO: 2:

-   -   Asp42 for Asn, Val, Ala or Ser    -   Ser92 for Ala, Gly, Val or Leu    -   Ser155 for Ala, Gly, Val or Leu    -   Glu311 for Gln, Asn, Leu, Ile or Met    -   Asp316 for Asn, Val, Ala or Ser    -   His320 for Asn, Phe, Asp or Tyr,    -   Gly322 for any amino acid larger than Asp such as Phe, Trp, Arg,        Lys, Glu, Gln, His, Tyr or Ile    -   Asp357 for Asn, Val, Ala or Ser    -   His358 for Asn, Phe, Asp or Tyr    -   His432 for Asn, Phe, Asp or Tyr

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Plasmid map of the expression vector pNaAP31-1 combing themutated gene sequence according to SEQ ID NO: 8 in pICZαA (Invitrogen).

FIG. 2: Plasmid map of the expression vector pNaAP31-1 combining themutated gene sequence according to SEQ ID NO: 8 in pIC9K (Invitrogen).

DETAILED DESCRIPTION OF THE INVENTION

A mutation of the amino acid sequence is understood as an exchange ofthe naturally occurring amino acid at the desired position for any otheramino acid which does not hinder folding into the correct tertiary andquaternary structure, but however, are not functional.

The sequence to be mutated (wild type) can for example be a humanintestinal AP or a human placental AP. In addition an AP of low activityor high activity from bovine intestine also come into consideration asthe wild-type AP according to the invention. All these enzymes are atleast 77% homologous to SEQ ID NO: 2. The homology was determined withthe software “Open VMS Vax Version V6.2”; (copyright) (c) 1982-2001,Genetics Computer Group, Inc.; A wholly owned subsidiary of OxfordMolecular Group, Inc. All rights reserved. Published research assistedby this software should cite: Wisconsin Package Version 10.2, GeneticsComputer Group (GCG), Madison, Wis.).

The stated position of the amino acid to be mutated relates to the aminoacid sequence of native alkaline phosphatase according to SEQ ID NO: 2,without a signal sequence. However, the stated amino acid positions arealso transferable to other bovine alkaline phosphatases or alkalinephosphatases from other organisms; the exchanges affect amino acids withhighly conserved functions within the protein and hence it is onlynecessary to adapt the position after the respective amino acidsequence.

Directed mutagenesis of the DNA sequence means that one or more codonsare changed by means of PCR mutagenesis. In this process only as fewchanges as necessary are changed in the base triplet of thecodon-optimized gene according to SEQ ID NO: 3 and in the mostfavourable case only one base is changed.

Mutants of the eukaryotic alkaline phosphatase are preferred accordingto the invention in which the mutant has one or more of the followingmutations and the position of the mutation is defined relative to theposition in SEQ ID NO: 2.

-   -   Asp42 for Val or Asn    -   Ser92 for Ala or Gly    -   Ser155 for Ala or Gly    -   Glu311 for Gln or Leu    -   Asp316 for Val or Asn    -   His320 for Asn or Phe    -   Gly322 for Phe or Lys    -   Asp357 for Val or Asn    -   His358 for Asn or Phe    -   His432 for Asn or Phe

The above-mentioned mutants of the eukaryotic alkaline phosphatase arepreferred according to the invention in which the enzyme having thewild-type sequence has a specific activity of more than 7000 U/mg. Inaddition a DNA sequence is preferred as a gene sequence which is basedon the gene which codes for a eukaryotic alkaline phosphatase mutanthaving a specific activity of more than 7000 U/mg and has beenspecifically mutated at a few positions such that the resulting DNAsequence codes for an amino acid sequence of the eukaryotic alkalinephosphatase mutant which is modified at one or a few positions, whereinthe mutation results in a substantial to total reduction of the specificactivity.

The mutants according to the invention of eukaryotic alkalinephosphatase are particularly preferred in which the mutant has an atleast 1000-fold reduced AP activity compared to the wild-type enzyme.Furthermore mutants of the eukaryotic alkaline phosphatase according tothe invention are preferred in which the mutant has an at least1000-fold reduced activity compared to the corresponding wild-typeenzyme. Those mutants are especially preferred according to theinvention in which the reduction of the specific activity is below thedetection limit (determined according to the activity test described inexample 4).

The following amino acid positions have proven to be suitable accordingto the invention: Asp316/His320/His432 (binding partners of zinc atom1), Asp42/Asp357/His358 (binding partners of zinc atom 2), Ser 55/Glu311(binding partners of the magnesium atom), Ser92 (hydroxyl group isdeprotonated for the nucleophilic attack on the substrate) (Ma andKantrowitz (1994), J. Biol. Chem. 16 pp. 31614-31619; Ma et al. (1995),Protein Science, 4, pp, 1498-1506; Kimura and Kikuta (2000) JBIC, 5, pp.139-155; Stec et al. (2000), JMB 299 pp. 1303-1311) and Gly322(important for the specific activity; EP 0 955 369). According to theinvention the positions described above come into consideration assingle mutants or also in all possible combinations of theabove-mentioned positions as double, triple or multiple mutants. Themutated amino acid sequences according to SEQ ID NO's: 4-7, areparticularly preferred in which SEQ ID NO: 4 represents a single mutantat position 92 (Ser92Ala), SEQ ID NO: 5 represents a single mutant atposition 322 (Gly322Phe), SEQ ID NO: 6 represents a double mutant atpositions 320 and 322 (His320Asn/Gly322Phe) and SEQ ID NO: 7 representsa triple mutant at positions 92, 320 and 322(Ser92Ala/His320Asn/Gly322Phe). The respective DNA sequences are shownin SEQ ID NO: 8-11.

The present invention also concerns a DNA which codes for the mutantaccording to the invention described above. Furthermore the presentinvention concerns vectors which contain the nucleic acid sequenceaccording to the invention. In particular these are vectors containing anucleic acid sequence, wherein the nucleic acid sequence is selectedfrom the SEQ ID NO's: 4-7. Suitable vectors are known to a personskilled in the art such as pPICZαA, B, C; pPICZ, pPICZ-E, pPICZα-E;pPIC6, pPIC6αA, B, C; pGAPZ, pGAPZαA, B, C; pPIC9; pPIC9K, pPIC3.5,pPIC3.5K, pAO815, pMET, pMETαA, B, C; pYES-DEST52, pYES2.1/V5-His-TOPO,pYC2-E, pYES2.1-E; YES vectors, pTEF1/Zeo, pTEF1/Bsd, pNMT-TOPO (e.g.Invitrogen). The corresponding gene sequences are for example clonedinto the vectors pPICZαA or pPIC9K which are commercially available(Invitrogen) and which contain the gene sequences of SEQ ID NO's: 8-11according to the invention which are under the control of the AOX1promoter. The following are examples of vectors prepared according tothe invention: vectors pNaAP31-1 (FIG. 1) and pNaAP31-2 (FIG. 2), whichhave the gene sequence according to SEQ ID NO: 8 each cloned intopPICZαA (pNaAP3′-1) and pPIC9K (pNaAP31-2).

The vectors pNaAP31-1 and pNaAP31-1 are equally relevant for the vectorsprepared according to the invention since the final production clone cancontain copies of both vectors.

An expression vector obtained according to the invention is preferablytransformed into various strains of yeast such as Pichia pastoris andstably integrated into the genome. An advantage of the stableintegration into the yeast genome is in particular that no selectionpressure is necessary when for example the weakly-active or inactivealkaline phosphatase mutants are subsequently produced in large volumefermentors. Stable integration into the genome means that the expressionvector is incorporated into the genome of for example Pichia pastoris bymeans of homologous recombination and is thus transmitted fromgeneration to generation as a permanent component of the yeast genome(Cregg, J. M. et al., Mol. Cell. Biol. 5 (1985), 3376-3385).

Another subject matter of the invention is a yeast strain transformedwith one of the vectors according to the invention. Methylotrophicyeasts such as the yeast Pichia pastoris, Hansenula polymorpha,Saccharomyces cerevisiae, Yarrowia lipolytica or Schizosaccharomycespombe are particularly suitable as the yeast host. Pichia pastoris isparticularly preferably used as the host strain. The Pichia pastorisX-33 strain is particularly preferably transformed with one of thedescribed vectors.

The invention also concerns a method for producing the mutant of theeukaryotic alkaline phosphatase according to the invention in yeastcells comprising the steps: a) cloning a gene sequence according to theinvention into different vectors, b) transforming the yeast, c)expression and d) purifying the alkaline phosphatase wherein

-   -   a first vector has a resistance gene to a first selection        marker,    -   transformants which have integrated the resistance gene and the        desired gene sequence into the genome are selected by growth on        nutrient medium containing a low concentration of a first        selection marker,    -   the gene copy number is increased by multiple transformation        whereby multiple transformants are selected by growth on        nutrient medium with increased selection pressure,    -   a second vector which has a resistance gene to a second        selection marker is added,    -   the gene copy number is increased by multiple transformation        whereby multiple transformants are selected by growth on        nutrient medium with increased selection pressure, and    -   those clones are selected which have stably integrated several        copies of the gene sequence and of the selection marker        resistance genes into the genome.

Methylotrophic yeast cells are preferably used in the method accordingto the invention. Pichia pastoris is particularly preferably used as theyeast cell.

Furthermore it is preferred that a vector is used for the methodaccording to the invention which essentially corresponds to a vectorwhich is selected from the following vectors: pPICZαA, B, C; pIZCZ,pPICZ-E, pPICZα-E, pPIC6, pPIC6αA, B, C; pGAPZ, pGAPZαA, B, C; pPIC9;pPIC9K, pPIC3.5, pPIC3.5 K, pAO815, (Invitrogen).

The copy number of the mutated gene sequence in the methylotrophic yeastwas increased by multiple transformation while simultaneously increasingthe selection pressure with a suitable selection marker e.g. anantibiotic such as Zeocin or GENETICIN (G418), after which only thoseclones are viable which have stably integrated several copies of theexpression vector into the genome. In order to be resistant to highconcentrations of the antibiotic used as the selection marker, it isnecessary that the clones produce increased amounts of resistanceproteins. This can for example be achieved by a multiple integration ofthe expression vector which, in addition to the expression cassette forthe respective AP mutant e.g. the alkaline phosphatase mutant Ser92Ala,also contains the resistance gene for the antibiotic used as theselection marker.

The object of producing alkaline phosphatase mutants in a robust andstable expression method having a high expression rate and at the sametime in economical yields was achieved by the method described in thefollowing.

The directed mutagenesis for producing the mutants according to theinvention was carried out as follows: based on the gene which codes fora bovine alkaline phosphatase mutant and which was preparedsynthetically, oligonucleotides which were complementary to one anotheror which overlapped were designed in which one or several base positionswere changed compared to SEQ ID NO: 3. One of these primers wassubsequently used in a PCR reaction as a partner of the 5′ or 3′ primerdescribed in the following and thus the AP gene was amplified in twosections containing the desired base exchange(s).

The two sections were subsequently analysed by means of agarose gelelectrophoresis, the products having the expected length were isolatedfrom the gel by means of the QIAquick gel extraction kit (QIAGEN) andsynthesized in a further PCR reaction to form the complete gene product.The first five cycles of the PCR reaction were carried out withoutadding the primer at the 5′ end and at the 3′ end of the whole gene sothat at first only a few fragments of the gene product of the expectedlength are formed from the two sections. The annealing temperaturedepends on the melting temperature of the overlapping region.Subsequently the terminal primers were added and the annealingtemperature was increased in accordance with the annealing temperatureof the primer with the lowest melting temperature. Afterwards the genefragment of the expected length was amplified in a further 25 cycles.

The PCR mixture was analysed by means of agarose gel electrophoresis andthe gene fragment having the expected size was isolated (QIAquick gelextraction kit/Qiagen).

The cloning of a corresponding PCR fragment, the transformation inPichia pastoris and the expression of the corresponding AP mutant aredescribed in example 2.

The recombinant alkaline phosphatase mutants can be isolated from thebiomass by extraction methods which are in principle known to a personskilled in the art e.g. by “protein purification”, Springer Publishers,Editor Robert Scopes (1982). A pure band product is achieved by suitablechromatographic methods such as in particular by using hydrophobiccolumn materials and a cation exchanger.

Hence the present invention describes for the first time a method whichallows an economical production of recombinant alkaline phosphatasemutants from mammals e.g. bovine intestine, in yeast which have a verygreatly reduced AP activity or no longer have an AP activity butotherwise have properties which correspond to those of recombinantalkaline phosphatase mutants from the bovine intestine. These mutantscan be used especially as interference-eliminating proteins inimmunological test procedures in which AP is used as a label such asMTP-ELISA.

Legends for the Sequence Protocols SEQ ID NO's 1-21

SEQ ID NO: 1: native DNA sequence coding for highly active bovine APwithout a signal sequence

SEQ ID NO: 2: amino acid sequence of the highly active bovine AP

SEQ ID NO: 3: DNA sequence of the synthetic gene coding for a highlyactive AP, the restriction cleavage site EcoRI is located upstream ofthe coding sequence and the restriction cleavage site Asp718 is locateddownstream thereof

SEQ ID NO: 4: amino acid sequence of the AP single mutant Ser92Ala(wild-type: highly active bovine AP)

SEQ ID NO: 5: amino acid sequence of the AP single mutant Gly322Phe(wild-type: highly active bovine AP)

SEQ ID NO: 6: amino acid sequence of the AP double mutantHis320/Gly322Phe (wild-type: highly active bovine AP)

SEQ ID NO: 7: amino acid sequence of the AP triple mutantSer92Ala/His320/Gly322Phe (wild-type: highly active bovine AP)

SEQ ID NO: 8: DNA sequence of the synthetic gene coding for the APsingle mutant Ser92Ala with the cleavage sites EcoRI and Asp718 locatedupstream and downstream respectively of the coding sequence

SEQ ID NO: 9: DNA sequence of the synthetic gene coding for the APsingle mutant Gly322Phe with the cleavage sites EcoRI and Asp718 locatedupstream and downstream respectively of the coding sequence

SEQ ID NO: 10: DNA sequence of the synthetic gene coding for the APdouble mutant His320Asn/Gly322Phe with the cleavage sites EcoRI andAsp718 located upstream and downstream respectively of the codingsequence

SEQ ID NO: 11: DNA sequence of the synthetic gene coding for the APtriple mutant Ser92Ala/His320Asn/Gly322Phe with the cleavage sites EcoRIand Asp718 located upstream and downstream respectively of the codingsequence

SEQ ID NO's: 12-21: DNA sequences which were used as primers

Abbreviations

-   -   YPD: yeast peptone dextrose    -   YPDS: yeast peptone dextrose sorbitol    -   BMGY: buffered glycerol-complex medium    -   BMMY: buffered methanol-complex medium    -   MTP: microtitre plates

SPECIFIC EMBODIMENTS Example 1

Mutagenesis of the Synthetic DNA Sequence which Codes for the BovineAlkaline Phosphatase Mutants

For the mutagenesis of the desired base triplet(s) by means of the PCRreaction, oligonucleotides were designed which have a correspondinglymodified base sequence and are complementary to one another or arepartially overlapping and complementary. These oligonucleotides weresubsequently used as corresponding partners for the 5′ primer 5′-hAPaccording to SEQ ID NO: 12 or for the 3′ primer 3′-hAP according to SEQID NO: 13 which each hybridize with the 5′ end or with the 3′ endrespectively of the gene which codes for the bovine alkaline phosphatasemutants. In this manner the mutated gene sequence was amplified in twosegments in a first reaction, the first segment carrying a mutation atthe 3′ end and the second segment carrying a mutation at the 5′ end anda short base sequence at the 3′ end of the first segment being identicalto a short base sequence at the 5′ end of the second segment.

These two segments were then fused in a second PCR reaction to form thefull length product. For this the PCR reaction was firstly startedwithout the 5′-hAP and 3′-hAP primers according to SEQ ID NO: 12 and 13and 5 cycles were carried out. A few molecules of the full lengthproduct are formed in this process; the annealing temperature in these 5cycles depends on the melting temperature of the overlapping region ofthe two segments. Subsequently the 5′ and 3′ primer according to SEQ IDNO: 12 and 13 are added, the annealing temperature is adapted to themelting temperature of the primer with the lower melting temperature andthe full length product is amplified in a further 25 cycles.

Mutagenesis to Generate the Single Mutant Ser92Ala

In order to generate the single mutant Ser92Ala the base triplet atposition 274-276, with reference to the base triplet TTG according toSEQ ID NO: 3, which codes for the first amino acid of the highly-activebovine alkaline phosphatase was mutated from TCT into GCT. The primers5′-S92A according to SEQ ID NO: 14 and 3′-S92A according to SEQ ID NO:15 which are partially complementary to one another were designed forthis purpose. The first PCR reaction was subsequently started with theprimer pairs 5′-hAP and 3′-S92A as well as 5′-S92A and 3′-hAP separatefrom one another using the gene sequence according to SEQ ID NO: 3 asthe template such that the mutated gene sequence was firstly amplifiedin two segments. The segments were analysed by an agarose gel andisolated from the agarose gel (QIAquick gel extraction kit/Qiagen) andsubsequently used in the second PCR reaction. In this second PCRreaction the two segments were fused as described above to form the fulllength product. The mutated gene sequence formed in this manner wascloned using PCR cloning vectors (PCR cloning kit—blunt end/RocheDiagnostics) and examined by means of restriction analysis andsequencing.

Mutagenesis to Generate the Single Mutant Gly322Phe

In order to generate the single mutant Gly322Phe the base triplet atposition 964-966, with reference to the base triplet TTG according toSEQ ID NO: 3, which codes for the first amino acid of the highly-activebovine alkaline phosphatase was mutated from GGT into TTT. The primers5′-G322F according to SEQ ID NO: 16 and 3′-G322F according to SEQ ID NO:17 which are partially complementary to one another were designed forthis purpose. The first PCR reaction was subsequently started with theprimer pairs 5′-hAP and 3′-G322F as well as 5′-G322F and 3′-hAP separatefrom one another using the gene sequence according to SEQ ID NO: 3 asthe template such that the mutated gene sequence was firstly amplifiedin two segments. The segments were analysed by an agarose gel andisolated from the agarose gel (QIAquick gel extraction kit/Qiagen) andsubsequently used in the second PCR reaction. In this second PCRreaction the two segments were fused as described above to form the fulllength product. The mutated gene sequence formed in this manner wascloned using PCR cloning vectors (PCR cloning kit—blunt end/RocheDiagnostics) and examined by means of restriction analysis andsequencing.

Mutagenesis to Generate the Double Mutant His320Asn/Gly322Phe

In order to generate the double mutant His320Asn/Gly322Phe, the basetriplets at positions 958-960 and 964-966, with reference to the firstbase triplet TTG according to SEQ ID NO: 3, which codes for the firstamino acid of the highly-active bovine alkaline phosphatase was mutatedfrom CAT into AAT and GGT into TTT. The primers 5′-H320N/G322F accordingto SEQ ID NO: 18 and 3′-H320N/G322F according to SEQ ID NO: 19 which arepartially complementary to one another were designed for this purpose.The first PCR reaction was subsequently started with the primer pairs5′-hAP and 3′-H320N/G322F as well as 5′-H320N/G322F and 3′-hAP separatefrom one another using the gene sequence according to SEQ ID NO: 3 asthe template such that the mutated gene sequence was firstly amplifiedin two segments. The segments were analysed by an agarose gel andisolated from the agarose gel (QIAquick gel extraction kit/Qiagen) andsubsequently used in the second PCR reaction. In this second PCRreaction the two segments were fused as described above to form the fulllength product. The mutated gene sequence formed in this manner wascloned using PCR cloning vectors (PCR cloning kit—blunt end/RocheDiagnostics) and examined by means of restriction analysis andsequencing.

Generation of the Triple Mutant Ser92Ala/His320Asn/Gly322Phe

The triple mutant Ser92Ala/His320Asn/Gly322Phe was generated bycombining the single mutant Ser92Ala and the double mutantHis320Asn/Gly322Phe. For this the two mutated gene sequences which wereeach cloned into PCR cloning vectors (PCR cloning kit—blunt end/RocheDiagnostics) were cleaved separately from one another with therestriction endonucleases MunI and Asp718, and the restriction mixturewas separated by means of agarose gel electrophoresis. MunI cleavesbetween the positions of the triplets of Ser92 and His320. A ca. 3700 bplong vector fragment was isolated from the single mutant Ser92Ala and aca. 900 bp long fragment of the 3′ region of the mutated gene sequenceof the double mutant His320Asn/Gly322Phe was isolated from the agarosegel and these two fragments were ligated together in the next step. Theresulting gene sequence was checked by sequencing.

Example 2

Cloning of the Mutated Gene Sequences into the Expression Vector pPICZαAfor Pichia pastoris

The verified mutated gene sequences were cleaved from the PCR cloningvectors by restriction with the restriction endonucleases EcoRI andAsp718, the restriction mixture was separated by means of agarose gelelectrophoresis and the ca. 1480 bp long fragments were isolated fromthe agarose gel (QIAquick gel extraction kit/Qiagen). Subsequently themutated gene sequences were ligated with the vector fragment from theexpression vector pPICZαA which was also linearized with EcoRI andAsp718. The restriction endonuclease cleavage sites required for EcoRIand Asp718 were incorporated into the mutated gene sequences by theprimers 5′-hAP and 3′-hAP which have recognition sequences for therestriction endonuclease EcoRI and Asp718 upstream and downstreamrespectively of the coding sequence. The resulting expression vectorsfor alkaline phosphatase mutants were designated as follows: pNaAP31-1(Ser92Ala), pNaAP43-1 (Gly322Phe), pNaAP51-1 (His320Asn/Gly322Phe) andpNaAP6-1 (Ser92Ala/His320Asn/Gly322Phe).

In this vector the mutated gene sequences are under the control of theAOX 1 promoter (promoter for alcohol oxidase 1 from Pichia pastoris)which can be induced with methanol and is cloned in the correct readingframe behind the signal peptide of the α factor from Saccharomycescerevisiae. The gene fragment inserted in this manner was then examinedby means of restriction analysis and sequencing for an error-free basesequence. The resulting expression vectors which each encode one of themutated gene sequences according to SEQ ID NO's.: 8-11, code foreukaryotic alkaline phosphatase mutants are shown with pNaAP31-1 as anexample (see FIG. 1).

Transformation of the Expression Vectors Containing the Mutated GeneSequences in Pichia pastoris

In order to transform the expression vectors containing mutated genesequences in Pichia pastoris X-33 with subsequent integration into thegenome, the vectors were firstly linearized with SacI (Roche DiagnosticsGmbH). The transformation was carried out by means of electroporationusing a Gene Pulser II (Biorad).

For this a colony of Pichia pastoris wild type strain was inoculated in5 ml YPD medium (Invitrogen) and incubated at 30° C. overnight whileshaking. The overnight culture was subsequently inoculated 1:2000 in 200ml fresh YPD medium (Invitrogen) and incubated overnight at 30° C. whileshaking until an OD₆₀₀ of 1.3-1.5 was reached. The cells werecentrifuged (1500×g/5 minutes) and the pellet was suspended in 200 mlice-cold sterile water (0° C.). The cells were again centrifuged(1500×g/5 minutes) and resuspended in 100 ml ice-cold, sterile water.The cells were again centrifuged and resuspended in 10 ml ice-cold (0°C.) 1 M sorbitol (ICN). The cells were again centrifuged and resuspendedin 0.5 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cells obtained inthis manner were kept on ice and used immediately for thetransformation.

About 1 μg linearized expression vector DNA was added to 80 μl of thecells and the entire mixture was transferred into an ice-cold (0° C.)electroporation cuvette and incubated for a further 5 minutes on ice.The cuvette was subsequently transferred to a Gene Pulser II (Biorad)and the transformation was carried out at 1 kV, 1 kΩ and 25 μF. Afterthe electroporation, 1 ml 1 M sorbitol (ICN) was added to the mixtureand subsequently 100 to 150 μl was plated out on a YPDS agar plate(Invitrogen) containing 100 μg/ml Zeocin (Invitrogen). The plates weresubsequently incubated for 2-4 days at 30° C.

The clones were inoculated onto raster MD (=minimal dextrose) plates andanalysed further. Grown clones were picked, resuspended in 20 μl sterilewater, lysed (1 hour, 37° C.) with 17.5 U lyticase (Roche DiagnosticsGmbH) and directly examined by means of PCR for the correct integrationof the expression cassette containing an appropriately mutated genesequence.

Clones which had integrated the complete expression cassette into thegenome in the transformation were then used in expression experiments.

Example 3 Expression of the Alkaline Phosphatase Mutants

Positive clones were inoculated in 3 ml BMGY medium (Invitrogen) andincubated overnight at 30° C. while shaking. Subsequently the OD at 600nm was determined and the inoculation in 10 ml BMMY medium (Invitrogen)was carried out in such a manner that it resulted in an OD₆₀₀ of 1. TheBMMY medium (Invitrogen) contains methanol (Mallinckrodt Baker B.V.)which induces the expression of the alkaline phosphatase mutants via theAOX 1 promoter.

The shaking flasks were incubated at 30° C. while shaking, samples wereremoved every 24 hours, the OD₆₀₀ was determined, an activity test wascarried out for the expression of the alkaline phosphatase mutants andeach time 0.5% methanol (Mallinckrodt Baker B.V.) was refed for furtherinduction. The expression experiments were carried out for 96 hours.

Example 4 Test for the Activity of Alkaline Phosphatase Mutants

500 μl aliquots of the expression culture of example 3 were removed, theOD₆₀₀ was determined and the cells were centrifuged. The supernatant wasstored and the cell pellet was resuspended in a quantity of Y-PER™ (50to 300 μl/Pierce) according to the OD₆₀₀ for lysis and shaken for 1 hourat room temperature. Subsequently the lysate was centrifuged in order toseparate the cell debris (15000×g/5 minutes) and the supernatant wastransferred to fresh reaction vessels. 5 μl of the lysate was then usedin the activity test.

The activity test functions according to the following principle:

The absorption increase at 405 nm is measured.

50 μl 4-nitrophenyl phosphate solution (0.67 mol/14-nitrophenylphosphate, Na salt (Roche Diagnostics GmbH) is added to 3 mldiethanolamine buffer (1 mol/l diethanolamine (Merck) pH 9.8, 0.5 mmol/lMgCl₂ (Riedel de Haen)) and the mixture was incubated at 37° C.Subsequently the reaction was started by adding 5 μl lysate and thechange in absorbance at 37° C. was determined for 3 minutes and theΔA/min was calculated from this.

The activity was then calculated according to the following formula:$\begin{matrix}{{activity} = {\frac{3.10}{\varepsilon \times 0.005 \times 1} \times \Delta\quad{A/\min} \times {\frac{1}{{factor}\quad x}\left\lbrack {U\text{/}{ml}\quad{sample}\quad{solution}} \right\rbrack}}} \\{\varepsilon = {18.2\quad\left\lbrack {1 \times {mmol}^{- 1} \times {cm}^{- 1}} \right\rbrack}} \\{{{factor}\quad x} = {{concentration}\quad{factor}\quad{after}\quad{cell}\quad{lysis}}}\end{matrix}$

The activity of the medium supernatant of the expression cultures wasdetermined in a similar manner. In this case the reaction was alsostarted with 50 μl supernatant but 0.5 mM ZnCl₂ was additionally added.The activity was then calculated without using factor x. Supernatants ofclones which express highly-active alkaline phosphatase according to SEQID NO: 3 were used as a positive control and clones which weretransformed with the initial vector pPICZαA without a target gene wereused as a negative control.

The residual activity of the mutants was determined with this activitytest as follows:

-   -   single mutant Ser92Ala: reduction of the specific activity by        ca. 5000-fold    -   single mutant Gly322Phe: reduction of the specific activity by        ca. 2500-fold    -   double mutant His320Asn/Gly322Phe: reduction of the specific        activity by ca. 10000-fold (near to the detection limit)    -   triple mutant Ser92Ala/His320Asn/Gly322Phe: reduction of the        specific activity by ca. 10000-fold (near to the detection        limit).

Example 5 Detection of the Expression of the AP Mutants by Western-Blot

10 μl unconcentrated supernatant or crude extract after cell lysis wasapplied to a 10% SDS gel (Novex Pre-Cast gel/Invitrogen) and theproteins that were present were separated according to size by applyingan electrical field. The proteins separated in this manner were blottedonto a nitrocellulose membrane (Novex Western Transfer Apparatus XCellII Blot Module/Invitrogen). After the blotting the membrane was washedtwice with 20 ml high-purity water for 5 minutes and subsequently shakenfor 30 minutes in 10 ml blocking solution (Invitrogen). The membrane wasthen again washed twice for 5 minutes with 20 ml high-purity water, andsubsequently incubated for 1 hour in 10 ml blocking solution(Invitrogen) which this time contained antibody 1(anti-AP-rabbit/Rockland Inc. diluted from a 10 mg/ml stock solution1:5000). It was then washed four times for 5 minutes each time with 20ml of an antibody wash solution (Invitrogen) and subsequently themembrane was incubated for 30 min with 10 ml of a secondary antibodysolution (contains the anti-rabbit antibody/Invitrogen). This wasfollowed by a four-fold wash for 5 minutes each time with 20 ml of anantibody wash solution (Invitrogen) and a three-fold wash with 20 mlhigh-purity water for 2 minutes each time. The membrane was thenincubated for 1-60 minutes with a dye solution (chromogenicsubstrate/Invitrogen) for the staining. The incubation period depends onthe quality of the stain. After optimal staining, the membrane is washedthree times with 20 ml high-purity water for 2 minutes each time andsubsequently the membrane is dried at room temperature. All solutionswith the exception of the high-purity water were derived from theWestern Breeze chromogenic immunodetection kit from Invitrogen, allincubation steps were carried out at room temperature. The procedure wasaccording to the instructions of the manufacturer.

Example 6 Increasing the Expression Rate by Multiple Transformation

The best clones from the expression experiments were in turn preparedfor electroporation as described in example 2 and again transformed with1 μg linearized expression vector vector DNA and the transformationmixture was plated out on YPDS agar plates (Invitrogen) containing 1000to 2000 μg/ml Zeocin (Invitrogen). In this manner the selection pressureis increased to such an extent that only those clones can grow whichhave integrated several copies of the expression vector and thus alsointegrated several copies of the respective resistance gene (in thiscase Zeocin) into the genome. The Zeocin resistance protein is theproduct of the bleomycin gene of Streptoalloteichus hindusstanus(Chalmels, T. et al., Curr. Genet. 20 (1991), 309-314; Drocourt, D. etal., Nucleic Acid Research 18 (1990), 4009), which binds Zeocin in astoichiometric concentration ratio and thus makes the cell resistant toZeocin. The higher the concentration of Zeocin in the YPDS agar plates,the more resistance protein the cell has to generate in order toquantitatively bind the Zeocin and thus enable growth. This is possiblewhen for example multiple copies of the resistance gene are integratedinto the genome. Clones were inoculated on raster MD plates as describedabove and again examined as described in example 2 by means of PCRanalysis for the correct integration of the respective expressioncassette. Subsequently these clones were again tested as described inexamples 4 and 5 for AP activity or by Western blot analysis.

Example 7 Increasing the Expression Rate by Using a Second SelectionPressure

Increasing the Zeocin concentration above 2000 μg/ml does not lead to animproved expression rate of the alkaline phosphatase mutants. The genecopy number of the genes according to SEQ ID NO's: 8-11 which code forthe alkaline phosphatase mutants and are codon-optimized for expressionin yeast was further increased in the expression clones by integratingadditional expression vectors into the genome of the expression clonefrom example 6 which had the highest expression rate which were selectedby means of a second selection pressure, preferably G418 (RocheDiagnostics GmbH). For this purpose the entire expression cassette frompNaAP31-1 consisting of AOX 1 promoter signal peptide of the α factorfrom Saccharomyces cerevisiae, codon-optimized gene for the alkalinephosphatase mutants according to SEQ ID NO's: 8-11 and AOX 1transcription termination region, was isolated by PCR usingappropriately selected primers and, as described below, integrated intothe vector pIC9K whose integration into the genome of Pichia pastoriswas selected by means of G418 (Roche Diagnostics GmbH). The primers usedin this case, 5′ expr and 3′ expr, have the sequences SEQ ID NO: 20 andSEQ ID NO: 21.

The PCR preparation was analysed by means of agarose gelelectrophoresis, the gene fragment having the expected size was isolated(QIAquick gel extraction kit/Qiagen), recleaved with SacI and NotI(Roche Diagnostics GmbH), subsequently isolated again from the agarosegel (QIAquick gel extraction kit/Qiagen) and ligated into a vectorfragment isolated from pPIC9K which had also been linearized withSacI/NotI (Roche Diagnostics GmbH). This ensures that the entireexpression cassette from the respective expression vectors was presentin an identical form in pPIC9K. The inserted fragment was examined bymeans of restriction analysis and sequencing with the flanking regions.The resulting expression vectors for alkaline phosphatase mutants weredesignated pNaAP31-2(Ser92Ala), pNaAP43-2 (Gly322Phe), pNaAP51-2(His320Asn/Gly322Phe) and pNaAP6-2(Ser92Ala/His320Asn/Gly322Phe).

The clones with the highest AP mutant expression rate from the multipletransformation using Zeocin as the selection marker were prepared forelectroporation as described in example 2 and transformed with 1 μgvector fragment pNaAP31-2 and derivatives linearized with SacI (RocheDiagnostics GmbH) as described in example 2. The transformationpreparation was subsequently stored for 1 to 3 days at 4° C. in 1 Msorbitol (ICN) (to develop the G418 resistance) and then 100 to 200 μlwas plated out on YPD plates (Invitrogen) containing 1, 2 or 4 mg/mlG418 (Roche Diagnostics GmbH) and incubated for 3 to 5 days at 30° C.The resulting clones were again examined as described above with theactivity test for an increased expression of the eukaryotic alkalinephosphatase mutants.

1. A recombinant DNA coding for a mutant of eukaryotic alkalinephosphatase, wherein said DNA comprises the nucleic acid sequence of SEQID NO:
 11. 2. A vector comprising a nucleic acid encoding an alkalinephosphatase, wherein said nucleic acid comprises SEQ ID NO:
 11. 3. Thevector of claim 2 wherein said vector is selected from the group ofvectors consisting of pPICZαA, B, C; pPICZ, pPICZ-E, pPICZα-E; pPIC6,pPIC6αA, B, C; pGAPZ, pGAPZαA, B, C; pPIC9; pPIC9K, pPIC3.5, pPIC3.5K,pAO815, pMET, pMETαA, B, C; pYES-DEST52, pYES2.1/V5-His-TOPO, pYC2-E,pYES2.1-E; YES-vectors, pTEF1/Zeo, pTEF1/Bsd, and pNMT-TOPO.
 4. A hostcell transformed with the vector of claim 2 or
 3. 5. The host cell ofclaim 4 wherein the cell is Pichia pastoris.
 6. A method for producing aeukaryotic alkaline phosphatase in yeast cells comprising the steps:preparing a first vector construct and a second vector construct whereinthe first and second vector constructs each comprise a recombinant DNAencoding the polypeptide of SEQ ID NO: 7, wherein the first vectorconstruct comprises a resistance gene to a first selection marker andthe second vector construct comprises a resistance gene to a secondselection marker, transforming said yeast cells with the first andsecond vector constructs, wherein said transforming comprises: selectingtransformants which have integrated DNA from the first vector constructinto a genome of the yeast cells by selected growth of the yeast cellson nutrient medium containing a low concentration of a first selectionmarker, increasing copy number of the integrated DNA from the firstvector construct by multiple transfections of the yeast cells andfurther selected growth of the yeast cells on nutrient medium containingincreased concentrations of a first selection marker, selectingtransformants which have integrated DNA from the second vector constructinto the genome of the host cell by selected growth of the yeast cellson nutrient medium containing a low concentration of a second selectionmarker, and increasing copy number of the integrated DNA from the secondvector construct by multiple transfections of the yeast cells andfurther selected growth of the yeast cells on nutrient medium containingincreased concentrations of a second selection marker, expressing thealkaline phosphatase, and purifying the alkaline phosphatase.
 7. Themethod of claim 6 wherein methylotrophic yeast cells are used.
 8. Themethod of claim 7 wherein Pichia pastoris is used as the yeast cell. 9.The method of claim 6 wherein the first and second vectors areindependently selected from the group of vectors consisting of: pPICZαA,B, C; pPICZ, pPICZ-E, pPICZα-E; pPIC6, pPIC6αA, B, C; pGAPZ, pGAPZαA, B,C; pPIC9; pPIC9K, pPIC3.5, pPIC3.5K, pAO815.
 10. A polynucleotideencoding the polypeptide of SEQ ID NO:
 7. 11. A vector comprising thepolynucleotide of claim
 10. 12. The vector of claim 11 wherein thevector is selected from the group consisting of pPICZαA, B, C; pPICZ,pPICZ-E, pPICZα-E; pPIC6, pPICαA, B, C; pGAPZαA, B, C; pPIC9; pPIC9K,pPIC3.5, pPIC3.5K, pAO815, pMET, pMETαA, B, C; pYES-DEST52,pYES2.1/V5-His-TOPO, pYC2-E, pYES2.1-E; YES-vectors, pTEF1/Zeo,pTEF1/Bsd, and pNMT-TOPO.
 13. A host cell transformed with the vector ofclaim 11 or
 12. 14. The host cell of claim 13 wherein the cell is Pichiapastoris.